Assessing the Public Health Implications of Virulent and Antibiotic-Resistant Bacteria in Côte d'Ivoire's Ready-to-Eat Salads

In Côte d'Ivoire, the popularity of ready-to-eat salads has grown substantially. Despite their convenience, these products often face criticism for their microbiological safety. This research was conducted to assess the virulence and antibiotic resistance profiles of Escherichia coli (E. coli), Salmonella spp., and Staphylococcus aureus (S. aureus) isolated from salads available in hypermarkets across Abidjan. The study utilized a combination of microbiological and molecular biology techniques. Results indicated that E. coli isolates harbored virulence genes such as stx2 (50%) and ST (62.50%), though genes stx1 and LT were absent in the samples tested. In S. aureus, virulence genes detected included sea (55.55%), sec (11.110%), and sed (44.44%). The antibiotic resistance assessment revealed high resistance in E. coli to β-lactam antibiotics, with all isolates resistant to cefuroxime (100%) and the majority to ampicillin and cefoxitin (87.5%). Most Salmonella spp. isolates were sensitive to the antibiotics tested, except for cefoxitin and ampicillin, showing resistance rates of 42.85% and 57.15%, respectively. Staphylococcus aureus demonstrated considerable resistance, particularly to cefoxitin (44.44%), benzylpenicillin (100%), and ampicillin (55.55%). In addition, resistance to aminoglycosides (55.55% to both kanamycin and gentamicin) and macrolides (66.66% to erythromycin and 55.55% to clindamycin) was noted. Resistance to various fluoroquinolones ranged between 33.33% and 55.55%. The presence of resistance genes such as blaTEM (10.52%), qnrA (2.26%), qnrB (5.26%), qnrS (5.26%), and mecA (13.15%) in E. coli and S. aureus underscores the challenge of multidrug resistance, exhibiting phenotypes such as ESBL (50%), Meti-R (55.55%), KTG (44.44%), MLSB (44.44%), and FQ-R (25%). These results carry significant epidemiological and public health implications, highlighting the urgent need for improved safety regulations and practices regarding ready-to-eat salads in urban food markets.


Introduction
Ready-to-eat fruit and vegetables, also known as 4th-range products, appeared in European supermarkets from 1980 onwards [1].According to WHO, FAO, and the World Cancer Research Fund, the consumption of 400-600 g of fruit and vegetables a day is recommended [2,3].A diet rich in fruits and vegetables is likely to reduce the risk of cardiovascular disease and protect against certain types of cancer [4].However, several cases of food poisoning associated with the consumption of these products have been observed around the world.For example, in 2012, a foodborne outbreak was reported in a college in China due to the consumption of salad ingredients [5].In May 2014, an outbreak was observed in military and civilian populations associated with the consumption of ready-to-eat mixed salad in Norway [6].In April 2019, a cross-border outbreak in Sweden including 37 cases and 20 cases in Denmark following consumption of imported fresh spinach was reported [7].Based on currently available statistics, bacterial species in this case, Escherichia coli (E.coli), Salmonella spp., and Staphylococcus aureus (S. aureus), are among the important pathogens associated with fourth-range foods [8].In Africa, the average prevalence of E. coli in ready-to-eat foods is 31.6%.Te prevalence of Salmonella spp. is estimated at 21.7% and that of S. aureus at 25.1% [9].Today, in developing countries such as Côte d'Ivoire, the consumption of readyto-eat salads has become a cause for concern.Indeed, the desire to eat well is now a general trend [10].However, high prevalences of E. coli (16%), Salmonella spp.(18%), and S. aureus (24%) have been determined in ready-to-eat salads sold in the city's supermarkets [11].Tus, this work was carried out to determine the levels of virulence and antibiotic resistance of E. coli, Salmonella spp., and S. aureus isolates isolated from ready-to-eat salads sold in Abidjan supermarkets.Tis work aims to prevent the dissemination of virulence and resistance genetic material of these pathogens through the consumption of these foods in Côte d'Ivoire.

Study Area.
Tis study examines industrially produced ready-to-eat salads available in large supermarkets within Abidjan, Côte d'Ivoire.Abidjan is located in the southern part of the country, along the Gulf of Guinea, and covers an area of 2119 km 2 , representing 0.6% of the national territory, with a population density of 1475 inhabitants per km 2 .Sample analyses were conducted in the laboratory at Université Jean Lorougnon Guédé in Daloa, the capital of the Haut-Sassandra region.Positioned centrally in the western part of Côte d'Ivoire, Daloa is situated at a latitude of 6 °52′38″ north and a longitude of 6 °27′00″ west, approximately 141 km from Yamoussoukro, the political capital and 383 km from Abidjan, the economic hub.Further studies on antibiotic resistance were carried out at the Bacteriology-Virology Department of the Institut Pasteur de Côte d'Ivoire, within the Antibiotics, Natural Substances, and Surveillance of Microorganisms and Antibiotics Unit (ASSURMI), located in Cocody.

Biological
Material.Tis experimental study analyzed bacterial isolates from 38 samples of ready-to-eat salads collected from hypermarkets in Abidjan, Côte d'Ivoire.Tese diferent samples come from 5 supermarkets located in the communes of Cocody (3) and Marcory (2).Te research focused on three bacterial species: Escherichia coli (8 isolates), Salmonella spp.(7 isolates), and Staphylococcus aureus (9 isolates).Tese isolates, previously characterized using conventional microbiological techniques, were sourced from a diverse range of salad types as detailed in Table 1.

Preparation of Genetic Material (DNA)
. DNA extraction was performed using the CTAB method, as outlined in reference [12].Te process began by centrifuging 1.5 mL of the bacterial preculture in LB medium at 16,000 rpm for 5 minutes to pellet the cells.After discarding the supernatant, the pellet was resuspended in 1.5 mL of CTAB1 extraction bufer (20 g/L CTAB, 1.4 mol/L NaCl, 0.1 mol/L Tris, 0.02 mol/L Na-EDTA, and a pH of 8.0) and 5 μL of RNAse (20 mg/mL).Tis mixture was then incubated at 60 °C for 30 minutes, with intermittent shaking to resuspend the material.Proteinase K (10 μL of 20 mg/mL) was added halfway through the incubation.
Following another round of centrifugation, 900 μL of the supernatant was extracted and mixed with an equal volume of chloroform.After vortexing and subsequent centrifugation at 15, 000g for 15 minutes, 650 μL of the clear supernatant was mixed with 1.3 mL of CTAB2 precipitation bufer and left to stand at room temperature for 60 minutes.Postcentrifugation, the supernatant was discarded, and the DNA pellet was washed with a NaCl solution (700 μL of CTAB3) followed by chloroform.Te aqueous phase was then mixed with twice its volume of cold isopropanol and allowed to precipitate at room temperature for 20 minutes.
Te DNA was then pelleted by centrifugation, washed with 70% ethanol, dried in an oven at 55 °C for 30 minutes, and fnally resuspended in 30 μL of TE bufer.Te extracted DNA was stored at −20 °C for further analysis.

Amplifcation of Desired Genes
. For E. coli, the target genes included those encoding Shiga toxins 1 and 2 (stx1 and stx2), which were amplifed using multiplex PCR.Te amplifcation also targeted genes for heat-labile (LT) and heat-stable (ST) toxins.Te PCR mix, based on the protocol described in [13], consisted of 20 μL total volume: 10 μL of 2X Phusion Master Mix, 2 μL of primers (both sense and antisense, detailed in Table 2), 2 μL of extracted DNA, and 6 μL of sterilized nuclease-free water.Te PCR conditions included an initial denaturation at 94 °C for 5 minutes, followed by 30 cycles of amplifcation (details in Table 3).Te PCR products, stained with 1 μL of 6X loading bufer, were analyzed on a 1.2% agarose gel.
For S. aureus, the study focused on characterizing fve stereotyped enterotoxins (sea, seb, sec, sed, and see), which are heat-stable proteins linked to food poisoning.Te virulence genes were amplifed in two multiplex sets: one for sea and seb and another for sec, sed, and see, using primers listed in Table 3. Amplifcations were conducted in a mini thermal cycler (miniPCR bio TM ), following the protocol in [15].Te resultant PCR products were subjected to electrophoresis on a 2% agarose gel.

Study of Antibiotic Resistance
2.4.1.Determination of Resistance Profle.Tis component of the study involved conducting antibiograms using the agar difusion method (Mueller Hinton, MH) as outlined by the antibiogram committee of the Société Française de Microbiologie [16].Initially, a 24-hour culture of each isolate was prepared.Subsequently, a bacterial suspension was made in a 2 mL solution of 0.85% NaCl to match the 0.   Plates were incubated within 15 minutes of disc placement, and the zones of inhibition around the antibiotic discs were measured using an automated system (ADAGIO).Te diameters of these inhibition zones were used to determine the sensitivity of the isolates, classifed as sensitive (S), resistant (R), or intermediate (I), according to the specifc criteria for each bacterium [16].

Detection of Genes Encoding Antibiotic Resistance.
Tis analysis included multiplex PCR amplifcation of betalactam resistance genes (blaTEM, blaSHV, and blaCTX-M) and fuoroquinolone resistance genes (qnrA, B, and S) in E. coli and Salmonella species.Te method used is that described by the authors in [41].Indeed, PCR reactions were performed using 2 μL of DNA template (density of 10 ng/ μL), 4 μL of Master Mix (5X), 1 μL of each primer (a total of 6 μL per multiplex), and 8 μL of H2O.Te reaction mix has a fnal volume of 20 μL.In addition, the mecA gene, responsible for meticillin resistance, was amplifed in staphylococci.Te primers for these resistance genes can be found in Table 5. Te reaction mixture used for this procedure was identical to that employed in the virulence gene testing.Specifc PCR conditions tailored to these resistance genes are detailed in Table 6.

Prevalences of Virulence Genes in Bacterial Isolates
Isolated from 4th-Range Salads.Te study identifed the presence of key virulence genes within isolates of S. aureus and E. coli isolated from ready-to-eat salads.For S. aureus, the sea gene showed a prevalence of 55.55%, detected in fve samples (SA, SCOM, SN, SChV, and SME).Te sec gene was less prevalent, found in only one sample (SCOM) at 11.11%.Te sed gene was identifed in four samples (Rq, SA, SCA, and SCOM), with a prevalence of 44.44%.
In E. coli, the virulence genes detected included ST and stx2, with prevalences of 62.50% (fve samples: SN, SCOM, SChV, SFR, and SME) and 50% (four samples: SN, SCOM, SChV, and SME), respectively.Detailed data on these fndings are available in Table 7.In addition, electrophoretic profles illustrating the amplifcation products of the sea and stx2 genes are presented in Figures 1 and 2, respectively.

Antibiotic Resistance Profle of Isolated
Isolates.In this study, the antibiotic resistance profles of bacterial isolates isolated from ready-to-eat salads were thoroughly    International Journal of Microbiology investigated.E. coli exhibited a high level of resistance, with all isolates (100%) resistant to cefuroxime, and a similarly high resistance was observed for cefoxitin and ampicillin at 87.5%.A lower resistance rate was noted for imipenem at 12.5%, while ciprofoxacin displayed a moderate resistance level of 37.5% (Table 8).For Salmonella spp., resistance to beta-lactam antibiotics was signifcant, with 42.85% of isolates resistant to cefoxitin and 57.15% to ampicillin.S. aureus isolates demonstrated extensive resistance across various antibiotics, with a complete resistance (100%) to benzylpenicillin and substantial resistance rates of 66.66% for both fosfomycin and erythromycin.In addition, resistance was noted at 55.55% for ampicillin, kanamycin, gentamicin, norfoxacin, and rifampicin, while 44.44% of isolates were resistant to cefoxitin and moxifoxacin (Table 8).Tese fndings underscore the critical challenge of combating antibiotic resistance in foodborne pathogens and highlight the need for stringent food safety regulations and proactive antibiotic stewardship.

Prevalence of Antibiotic Resistance Genes and Phenotypes in Isolated Isolates.
A comprehensive analysis of resistance genes in E. coli, Salmonella, and S. aureus isolated from ready-to-eat salads was conducted.Tus, the signifcant resistance of the E. coli species to ticarcillin + clavulanic acid (TCC) and cephalosporin (cefoxitin, cefxime, and cefuroxime) results in the ESBL phenotype (50% of isolates).However, 10.52% of beta-lactam resistant species tested positive for the blaTEM resistance gene.Fluoroquinolone resistance (FQ-R) was identifed through the presence of qnrA (2.26%), qnrB (5.26%), and qnrS (5.26%).Te presence of resistance genes in E. coli isolates reveals high resistance to fuoroquinolones (25%).No resistance genes were detected in Salmonella isolates.However, most S. aureus isolates (55.55%, or 5 out of 9) harbored the mecA gene, indicative of the Meti-R phenotype, refecting meticillin resistance.In addition, resistance to aminoglycosides was observed in 55.55% of S. aureus isolates for both gentamicin and kanamycin, contributing to a KTG phenotype afecting 44.44% of these isolates.Te MLSB phenotype, linked to resistance to macrolide-lincosamide-streptogramin B antibiotics such as clindamycin (55.55%) and erythromycin (66.66%), was also prevalent in 44.44% of the S. aureus isolates.
Tese fndings on the prevalence of antibiotic resistance genes and corresponding phenotypes are depicted in Figure 3 for genes and Figure 4 for phenotypes.
3.2.Discussion.Te analysis of virulence and antibiotic resistance in bacterial isolates isolated from ready-to-eat salads sold in supermarkets in Abidjan revealed notable fndings.In E. coli isolates, the presence of stx2 genes was detected in four samples, demonstrating a prevalence of 50%.In addition, the ST gene was found in fve samples, corresponding to a 62.50% prevalence.Notably, neither stx1 nor LT genes were identifed in any of the samples.
Among the four virulence genes (stx1, stx2, ST, and LT) studied, only stx2 was linked to Shiga toxin (STEC) production.A study in the United States found similar trends where the stx1 gene was absent in tested isolates, whereas 93.1% carried the stx2 gene [23].Moreover, an investigation in Iran revealed a high prevalence of stx2 genes in cattle feces, supporting the potential for fecal contamination in agricultural settings [13].
Regarding the toxins associated with thermolabile (LT) and thermostable (ST) toxins in E. coli, only the ST gene was found, detected in fve samples representing a prevalence of 62.50%.Comparable research conducted in Casablanca identifed the ST gene in certain food products, suggesting possible fecal contamination through irrigation water used in market gardening [24].
Analysis of S. aureus in this study revealed the presence of the sea, sec, and sed genes with prevalences of 55.55%, 11.11%, and 44.44%, respectively.Te results of another study showed the absence of enterotoxin genes in readyto-eat salads, suggesting signifcantly better hygiene conditions in the production of 4th-range salads [25] than this.In addition, the literature suggests that the presence of certain preservatives such as lactic acid, produced by bacteria, can inhibit SE formation in foods, potentially via an extracellular protease, leading to a decrease in enterotoxin levels under specifc conditions [29].In this context, the detection of enterotoxin A, C, and D genes in S. aureus in the salads analyzed could indicate defciencies in personnel hygiene, potentially involving healthy carriers of these genes.
Te phenotypic characterization performed on food pathogens isolated from ready-to-eat salads sold in hypermarkets in Abidjan highlights signifcant concerns, 6 International Journal of Microbiology particularly the presence of multiresistant bacteria.Tese pathogens, which survive without heat treatment in 4th-range foods, pose severe health risks, potentially leading to fatal outcomes for consumers.During this study, antibiotic susceptibility testing revealed a pronounced resistance in E. coli to beta-lactam antibiotics, with 100% resistance to cefuroxime (CXM) and 87.5% resistance to both ampicillin (AMP) and cefoxitin (FOX).Similarly, Salmonella spp.showed resistance rates of 42.85% to cefoxitin (FOX) and 57.14% to ampicillin (AMP).Tese fndings align with those from other regions;  International Journal of Microbiology for example, a study in northern California found 76% of E. coli isolates resistant to ampicillin and lower resistance to cefoxitin (23%) and cefuroxime (20%) [26].Contrastingly, a study in southwestern Nigeria reported a 65.7% resistance rate of E. coli to cefuroxime [27], while a diferent study in Abidjan found no resistance in E. coli to cefuroxime [28], suggesting variability in bacterial resistance based on the source of salad ingredients.Moreover, resistance to fuoroquinolones was also noted, closely mirroring results from other studies on ready-to-eat foods, with 61.29% resistance to ciprofoxacin observed [29].Similar research found resistance rates to ciprofoxacin at 8.3% and to carbapenems at 5% [30].However, this study found a low resistance in E. coli to imipenem at 2.9%, akin to fndings by another study [31].
For Salmonella spp., the observed resistance profle in this study aligns with the signifcant resistance to ampicillin reported elsewhere, such as 88% in S. enteritidis [32] and 89.9% in Nigerian samples [33].Additional work on retail meat revealed a notable resistance in Salmonella serotypes to ampicillin at 29% and to cefoxitin at 30.43%, further supporting the patterns of resistance found in this study [34].Antibiotic susceptibility testing of S. aureus isolates revealed signifcant resistance levels to various antibiotic families.High resistance rates were observed with the beta-lactam antibiotics, notably cephalosporins such as cefoxitin (44.44%) and penicillins, with a 100% resistance to benzylpenicillin and 55.55% to ampicillin.For aminoglycosides, resistance levels were 55.55% for both kanamycin (KMN) and gentamicin (GMN).Resistance to macrolides was also substantial, with erythromycin (ERY) at 66.66% and clindamycin (CMN) at 55.55%.Resistance to fuoroquinolones varied, peaking at 55.55% for norfoxacin (NXN).Resistance to beta-lactam antibiotics in S. aureus can be attributed to the production of beta-lactamase, which hydrolyzes the beta-lactam ring of penicillins or intrinsic resistance mechanisms such as modifcation of penicillin-binding proteins (PBPs) or acquisition of new PBPs.Tis phenomenon of meticillin resistance, which leads to broadspectrum resistance to all beta-lactam antibiotics, is described in references [35,36].Macrolide resistance generally involves the action of a methylase enzyme that modifes the 23S subunit of ribosomal RNA.Comparatively, these resistance patterns correspond closely to results obtained in Côte d'Ivoire, where one study documented a 50% resistance rate to erythromycin [37].
Fluoroquinolone resistance often involves three primary mechanisms: target modifcation through mutations in the grlA or grlB genes of topoisomerase IV [38].Other significant resistance observed included fosfomycin (66.66%), rifampicin (50%), and chloramphenicol (37%).A study conducted in Greece by the authors in [39] explored the prevalence, distribution, and antibiotic susceptibility of S. aureus in ready-to-eat salads and environmental and personnel samples from a salad production facility.Tey found S. aureus in 27% of samples, with most isolates showing resistance to 2-5 antibiotics, including fosfomycin.Tis aligns with the fndings in the current study, which showed higher resistance rates to fosfomycin (82.6%), rifampicin (55.55%), and chloramphenicol (44.44%), suggesting a more pronounced resistance profle compared to the Greek study.
During this study, the assessment of antibiotic resistance profles necessitated identifying specifc resistance genes in E. coli, Salmonella, and S. aureus isolates from ready-to-eat salads.E. coli resistance to beta-lactam antibiotics in this study was high, with a 50% ESBL (extended-spectrum betalactamase) phenotype.However, the blaTEM gene was only identifed in 10.52% of isolates.In addition, fuoroquinolone resistance was evident via the qnrA gene (2.26%),In addition, resistance to fuoroquinolones was evident through the qnrA (2.26%), qnrB (5.26%), and qnrS (5.26%) genes, which collectively underpinned fuoroquinolone resistance in 25% of cases.Tese fndings align with those reported in [40].In S. aureus, the mecA gene was detected with a prevalence of 13.15%, accounting for 55.55% of the isolates and indicating resistance to meticillin, characteristic of the Meti-R phenotype.Te study also found signifcant resistance to aminoglycosides, specifcally gentamycin and kanamycin, each at 55.55%, correlating with the KTG phenotype in 44.44% of the samples.In addition, high rates of resistance to clindamycin (55.55%) and erythromycin (66.66%) were observed, contributing to the MLSB phenotype in 44.44% of the isolates.A study cited as [39] noted that in MRSA isolates, resistance rates exceed 90%, mirroring the fndings of this study where all identifed multiresistant isolates exhibited four resistance phenotypes: Meti-R, KTG, MLSB, and resistance to fuoroquinolones.Such elevated resistance levels could signifcantly contribute to the rise in nosocomial infections and complicate adherence to antibiotic therapy protocols by both patients and healthcare providers.Ultimately, this situation underscores the critical need for standardized antibiogram practices to guide efective antibiotic treatments, especially in cases where standard protocols may not be adequately followed.

Conclusion
Te study of virulence and antibiotic resistance in bacterial isolates isolated from ready-to-eat salads has underscored signifcant health risks to consumers.Particularly, the pathogenic potential of E. coli was demonstrated through the identifcation of stx2 and ST genes with prevalences of 50% and 62.50%, respectively.Similarly, the detection of sea, sec, and sed genes in S. aureus isolates, having prevalences of 55.55%, 11.11%, and 44.44%, respectively, highlights the virulent nature of these pathogens.Te antibiotic resistance profles further reveal that isolates of S. aureus and E. coli exhibit multiple resistances to antibiotics, complicating treatment options.Notably, E. coli isolates showed substantial multiresistance, evidenced by the presence of bla-TEM genes (10.52% prevalence), which contribute to an ESBL phenotype in 50% of the isolates.In addition, the detection of qnrA (2.26%), qnrB (5.26%), and qnrS (5.26%) genes indicates a 25% prevalence of resistance to quinolones.S. aureus isolates displayed resistance to meticillin, as marked by the mecA gene (13.15%prevalence) and a Meti-R rate of 55.55%.High resistance rates were also observed in aminosides and macrolides, characterized by KTG and MLSB phenotypes, both at 44.44%.Tese fndings suggest that the consumption of contaminated ready-to-eat salads could lead to therapeutic challenges in treating human infections due to the high prevalence of antibiotic-resistant bacteria.As these salads become a more common part of the diet, it is imperative to enforce stringent safety measures and monitoring to mitigate the risk of consumer poisoning and limit the spread of multiresistant bacteria.Tis approach is crucial in preserving the efcacy of antibiotics and ensuring the health and safety of consumers.

Table 1 :
Detection of pathogenic bacteria in ready-to-eat salads.

Table 4 :
List of antibiotic discs tested on bacterial isolates.

Table 7 :
Prevalence of virulence genes in E. coli and S. aureus.

Table 8 :
Resistance profle of bacterial isolates isolated from ready-to-eat salads.